A noncanonical function of EIF4E limits ALDH1B1 activity and increases susceptibility to ferroptosis

Ferroptosis is a type of lipid peroxidation-dependent cell death that is emerging as a therapeutic target for cancer. However, the mechanisms of ferroptosis during the generation and detoxification of lipid peroxidation products remain rather poorly defined. Here, we report an unexpected role for the eukaryotic translation initiation factor EIF4E as a determinant of ferroptotic sensitivity by controlling lipid peroxidation. A drug screening identified 4EGI-1 and 4E1RCat (previously known as EIF4E-EIF4G1 interaction inhibitors) as powerful inhibitors of ferroptosis. Genetic and functional studies showed that EIF4E (but not EIF4G1) promotes ferroptosis in a translation-independent manner. Using mass spectrometry and subsequent protein-protein interaction analysis, we identified EIF4E as an endogenous repressor of ALDH1B1 in mitochondria. ALDH1B1 belongs to the family of aldehyde dehydrogenases and may metabolize the aldehyde substrate 4-hydroxynonenal (4HNE) at high concentrations. Supraphysiological levels of 4HNE triggered ferroptosis, while low concentrations of 4HNE increased the cell susceptibility to classical ferroptosis inducers by activating the NOX1 pathway. Accordingly, EIF4E-dependent ALDH1B1 inhibition enhanced the anticancer activity of ferroptosis inducers in vitro and in vivo. Our results support a key function of EIF4E in orchestrating lipid peroxidation to ignite ferroptosis.

(c) Cell viability of control and EIF4E-overexpressing (EIF4E OE ) HT-1080 and Calu-1 cells following treatment with RSL3 or erastin at the indicated dose for 24 hours (n = 3 biologically independent samples; data are presented as mean ± SD).
(d) Cell death of indicated cells following treatment with RSL3 (0.5 μM) or staurosporine (0.25 μM) for 24 hours (n = 3 biologically independent samples; two-way ANOVA with Tukey's multiple comparisons test; data are presented as mean ± SD).  (c) Western blot analysis of the indicated proteins in control and EIF4G1 knockdown (EIF4G1 KD ) HT-1080 and Calu-1 cells. 4 (d and e) Propidium iodide (PI) staining analysis of cell death in control and EIF4G1 knockdown (EIF4G1 KD ) HT-1080 and Calu-1 cells following treatment with 0.5 μM RSL3 (B) or 5 μM erastin (C) for 24 hours (n = 3 biologically independent samples; data are presented as mean ± SD).   6 (e) Western blot analysis of the indicated proteins in wide type (WT) and EIF2S1 S51A U2OS cells following treatment with RSL3 (0.5 μM) for 6 hours.
(f) Cell death analysis of indicated U2OS cells following treatment with RSL3 for 24 hours (n = 3 biologically independent samples; data are presented as mean ± SD).
(g) HT-1080 cells were transfected with a dual luciferase reporter plasmid including HCV IRES activity linked to renilla luciferase (REN) and cap-dependent translation activity linked to firefly luciferase (FF). The transfected cells were treated with 4EGI-1 (10 μM) or 4E1RCat (10 μM) and harvested for luciferase assay (RLA; n = 3 biologically independent samples; data are presented as mean ± SD). data are presented as mean ± SD).

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(c) qPCR analysis of ALDH1B1 and ELF4E expression in indicated Calu-1 cells (n = 3 biologically independent samples; data are presented as mean ± SD).
(d) Cell viability of indicated Calu-1 cells following treatment with RSL3 or erastin at the indicated dose for 24 hours (n = 3 biologically independent samples; data are presented as mean ± SD).
(e) Cell viability assay of indicated Pfa1 cells after knockout of Gpx4 for 24 hours (n = 3 biologically independent samples; data are presented as mean ± SD).   (a) Athymic nude mice were injected subcutaneously with indicated HT-1080 cells for 7 days and then treated with IKE (40 mg/kg, i.p., once every other day) at day 7 for 2 weeks. Tumor volumes were calculated weekly (n = 6 mice/group; two-way ANOVA with Tukey's multiple comparisons test; data are presented as mean ± SD).

(b) Photographs of isolated tumors at day 14 after treatment.
(c-f) The levels of 4HNE (c), PTGS2 mRNA (d), EIF4E protein (e), and NOX1 protein (f) in isolated tumors at day 14 after treatment were assayed (n = 6 mice/group; two-way ANOVA with Tukey's multiple comparisons test; data are presented as mean ± SD).
(g) Athymic nude mice were injected subcutaneously with indicated HT-1080 cells for 7 days and then treated with IKE (40 mg/kg, i.p., once every other day) at day 7 for 2 weeks. Tumor volumes were calculated weekly (n = 6 mice/group; two-way ANOVA with Tukey's multiple comparisons test; data are presented as mean ± SD).
(h) Photographs of isolated tumors at day 14 after treatment.
(i-l) The levels of 4HNE (i), PTGS2 mRNA (j), EIF4E protein (k), and NOX1 protein (l) in isolated tumors at day 14 after treatment were assayed (n = 6 mice/group; two-way ANOVA with Tukey's multiple comparisons test; data are presented as mean ± SD).